Method and apparatus for collecting a cell sample from a liquid specimen

ABSTRACT

A method and apparatus for collecting a cell sample from a liquid specimen utilizes a collection receptacle in which the specimen is deposited, with a filter media being place in communication with a discharge port in the collection receptacle and a transfer device for drawing specimen from the collection receptacle and through the filter for capturing the cellular component of the specimen on the filter media for defining a cell sample for analysis. The collection receptacle, filter media and carrier therefor and the transfer device may be supplied in kit form for use in a clinical environment. A hypobaric vessel may be used and the transfer device and this vessel may also serve as a disposal receptacle for the liquid specimen passed through the filter media.

The invention is generally related to an apparatus for isolatingcellular samples from liquid specimens such that the cells are availablefor subsequent analysis. More specifically, the invention is directed toa device for collecting high-volume liquid specimens, such as urine ormouthwashes, and separating the cellular components from the liquidcomponent, the cellular component being used for analysis, includingmicroscopy and DNA analysis.

BACKGROUND OF THE INVENTION

Collection of liquid specimens for laboratory analysis in order todetect the presence of specific diseases or conditions in a patient iswell known. Typically, a liquid specimen or a swab is collected and,depending on the desired assay, the appropriate component of thespecimen is extracted. In cases where the desired component is cellularor subcellular, the specimens are generally centrifuged to pellet thecells. The cell pellets are optionally lysed to release a subcellularcomponent. Alternatively, lysis may occur prior to centrifugation andthe pelleted debris can be analyzed. Because centrifugation equipment isnot readily portable, specimen collection, especially high-volume liquidspecimen collections, have generally been limited to the clinical orlaboratory setting. While swabs have been transported for years, thecollection process generally requires trained technicians to assure thecollection of a useful specimen and careful storage and transport ofresuspended cells from the swab.

More recently, mouthwash specimens have been introduced as a method ofcollecting patient cell specimens for analysis. Typically, a patient isgiven an oral rinse or mouthwash which is expectorated into a collectioncontainer upon completion of the rinse step. The resulting mouthwashspecimen contains saliva and sloughed buccal cells mixed in with theexpectorated rinse. Such a mouthwash specimen can be analyzed todetermine its various components or certain patient attributes.

For example, as reported in The Lancet, Vol. 340, Jul. 25, 1992, pp214-216, a method of collecting mouthwash specimens to collect patientcells for cystic fibrosis screening has been studied in the UnitedKingdom. Cellmark Diagnostics has also developed a method for extractionof DNA from mouthwash specimens as part of its CF Mutation AnalysisSystem. In the Cellmark process, once the mouthwash specimen iscollected, it is centrifuged and the desired components are extractedfrom the pelleted cells.

The centrifugation step is normally done at the collection site. Inorder to perform the collection and analysis using this system, thecollection of the specimen must be at a site where equipment isavailable for centrifugation and extraction. In addition, only a smallpercentage of the total specimen is required for tests. This means thatsince the entire specimen must be kept viable, the entire specimen mustbe stored until the extraction step is completed. In a typical example,only one percent of the specimen is required for an analysis. This means99% of the storage specimen is ultimately discarded. By maintaining sucha large specimen, the costs of transportation, storage and disposal ofthe specimen becomes critical.

While mouthwash collection has been found to be an efficient anddesirable, non-invasive method of collecting cell specimens from a humanpatient, the transportation, storage and disposal problems haveminimized its widespread acceptability. In addition, the fact that theprimary cell collection steps must be performed at a central locationfurther diminish the value of this method of collection.

In order to analyze mouthwash specimens properly, the specimens must becollected in fluid form in a sterile container, sealed and transportedto the central centrifugation and extraction site. This is also true ofmost other collected liquid specimens of bodily fluids.

Therefore, the advantages of specimen collection are often outweighed bythe disadvantages of the cumbersome, inconvenient and costly stepsrequired in the storage preservation and transportation of the specimento an analysis site. In order for the liquid specimen techniques to gainwidespread acceptance, a need must be met to minimize the difficulty incollecting, storing and transferring the specimens obtainable from theliquid solution.

For example, U.S. Pat. No. 3,888,629, entitled: Performance of Chemicalor Biological Reactions within Absorbent Matrix Pad, issued to K. D.Bagshawe on Jun. 10, 1975 shows a system for drawing a liquid specimenthrough a discrete matrix pad for analysis of an antigenic component. Itdoes not, however, deal with the collection of the original specimen orwith the isolation of cells for analysis. This is also true with thedevice shown and described in U.S. Pat. No. 4,891,134, entitled:Specimen Filtration Device, issued to J. Vcelka on Jan. 2, 1990. In bothof these documents the cells are lysed to release the antigeniccomponent of interest prior to application to the matrix filter.

While numerous examples are available for introducing a specimen to atest medium such as a matrix pad or the like, the clinical use of suchsystems has generally been limited because of the requirement for theentire liquid test specimen to be transported in a liquid state from thecollection site to the analysis facility, thereby requiring sealed,sterile containers and shipping parcels which must be handled with greatcare. This is true whether the specimen is generated using invasivetechniques (such as the collection of blood specimens) or in annon-invasive manner (such as urine or mouthwash specimens). Therefore,there remains a need to improve the clinical environment for thecollection and transportation of liquid specimens.

The so-called "Guthrie Spot" is universally used for screening neonatalwhole blood for a variety of products of errors of metabolism {R.Guthrie, Organization of a regional newborn screening laboratory, inNeonatal screening for inborn error of metabolism (ed. H. Bickel, R.Guthrie and G Hammersen), pp 259-270, Springer Verlag, Berlin 1980}. Thedried blood spots are of great utilty because they facilitate theability to ship, archive and perform multiple analyses on the samesample. More recently, the utility of such dried blood spots has beenextended to tests involving DNA amplification and analysis (McCabe ERB.1991. Utility of PCR for DNA Analysis from Dried Blood Spots on FilterPaper Blotters, in PCR Methods and Applications, Volume 1:pp 99-106).Application of the technique is limited, however, and has only beenapplied to analysis of blood samples. Moreover, it does not involve anyseparation of cells from a liquid component. Both cells and serum remainon the filter. The present invention, as will be seen, extends theutility and obviates these disadvantages of the Guthrie Spot.

SUMMARY OF THE INVENTION

The subject invention is directed to a method and apparatus forexpediting the collection and transfer of cellular components of bodilyfluids collected as a liquid specimen. The invention is particularlyuseful in the isolation of cellular components of high-volume liquidspecimens, especially those collected by way of non-invasive processes,such as by way of example, buccal cells which may be collected from amouthwash specimen, or pathogen cells which may be found in urine.However, it will be understood that the invention can also be utilizedfor collection and analysis of other liquid specimens collected throughinvasive techniques, such as blood or CSF specimens and the like.

Thus, in a first aspect, the invention relates to a cell collectionapparatus for collecting, archiving and transporting a cell samplegenerated from a liquid specimen, comprising:

a liquid specimen collection container having an open end for receivinga liquid specimen and a discharge port through which said liquidspecimen can leave the collection container;

a filter container having an inlet port adapted for sealably engagingthe discharge port of said collection container, an outlet port throughwhich said liquid specimen can leave the filter container and a filtermedia supported in the container between said inlet and outlet ports,whereby liquid specimen entering said filter container via the inletport passes through said filter media before leaving via said outletport, said filter media being adapted for receiving and capturing acellular component of the liquid specimen for defining a cell sample foranalysis; and

means for creating and applying a reduced pressure to the outlet port ofsaid filter container, thereby drawing the liquid specimen through thefilter media.

Preferably the cell collection apparatus further comprises a dischargevessel for receiving the liquid specimen passing through the filtermedia and the outlet port of the filter container; and more preferablythe means for creating and applying reduced pressure is a hypobaricvessel adapted to be coupled in sealed engagement with the outlet portof the filter container. The hypobaric vessel can thus simultaneouslyserve as the means for applying pressure and the discharge vessel. AVacutainer™ vessel has been found convenient and suitable for a combineddischarge vessel and means for applying reduced pressure in accordancewith the present invention.

The cell collection apparatus may also include a hollow-bore needle incommunication with said filter container outlet port, said needle beingadapted for piercing a rupturable seal on said hypobaric vessel, therebyapplying a reduced pressure to the outlet port.

Preferably, the filter media is a discrete element, all or part of whichis removable from said filter container. It may be made of a materialselected from the group consisting of glass fiber, paper and celluloseesters. To facilitate removal of the filter, the filter containerhousing may comprise a pair of mated members. In such case, the onemember may include a filter support means for supporting the filtermedia. The filter housing preferably also includes channeling means fordistributing liquid specimen to discrete areas of the filter media.

In another aspect, the invention relates to methods of collectingcellular samples from liquid specimens, the method comprising the stepsof:

depositing a liquid specimen suspected of having a cellular componentinto an open end of a collection receptacle having a discharge port;

placing the discharge port in fluid communication with a filter media bymeans of a sealed passage therebetween, the filter media having a poresize sufficiently small to retain desired suspected cells from thespecimen, the; and

applying a pressure differential across the filter and extending to thecollected specimen by means of the sealed passage to force the liquidspecimen through the discharge port of the collection receptacle andthrough the filter media, thereby capturing on the filter media anycellular component of the liquid specimen.

The pressure differential may be created by placing the outlet port ofthe filter container under a relative vacuum, such as is accomplished byplacing a closed hypobaric chamber in fluid communication with saidoutlet port. Typically, the hypobaric chamber includes a penetrableclosure element, and is selectively placed in fluid communication withsaid outlet port by mounting a hollow bore needle to said filtercontainer such that the bore of the needle is in fluid communicationwith the outlet port, and puncturing the closure element with theneedle, thereby placing the filter container under hypobaric pressure.

Following collection of cells in the above manner, at least some of thefilter media may be removed from the filter container and the sampledried on the filter media. Removal can be by opening of mated halves ofthe filter container housing, or by punching portions of the filtermedia to the exterior of the housing. The cell sample can then besectioned, stored, transported and/or analyzed according to the wishesof the clinician.

While the cell sample can be analyzed for any number of consitutents, byany number of known analytical techniques, a preferred constituent foranalysis is a segment of nucleic acid, preferably the RNA or DNA fromthe cells. Such nucleic acid may be analyzed in situ or byamplification, such as by PCR or LCR.

It is presently preferred that the liquid specimen is a high-volumespecimen, such as a mouthwash specimen.

In a further aspect, the invention relates to kits containing thecomponents of the apparatus for practicing the above methods.

The subject invention permits the rapid collection of cells on a filtermedia in a manner that can be performed at any site without specialequipment or special training, producing a very portable andtransportable filter paper specimen which can be dried and archived forone or more subsequent analyses. The cell collection method andapparatus of the subject invention has an unexpected desired result inthat the collection of buccal cells quickly after the mouthwash specimenhas been produced has provided unusually high and consistent yield ofcells in each collected sample.

The invention provides a desirable method and apparatus for use in anyapplication where liquid specimens are collected for analysis of thecellular component thereof, and has proven to be particularly useful inDNA amplification technology such as for the investigation of geneticdefects, carrier status for genetic defects, as well as forensic,identity, military or paternity applications.

The invention is readily adapted to be supplied to clinical techniciansin kit form with an integrated collection receptacle, filter media packand hypobaric vessel. The method of specimen collection and samplecapture is quickly mastered without specialized training andconsistently yields excellent samples for analysis. The components ofthe kit are inexpensive and their use is consistent with widely known,proven laboratory techniques.

It is, therefore, an object and feature of the subject invention toprovide specimen collection apparatus for collecting a cell rich samplefor archiving, transporting and subsequent analysis.

It is another object and feature of the subject invention to provide aspecimen collection device for collecting human cells on a filter mediaby drawing the specimen through the filter.

It is also an object and feature of the subject invention to provide asample collection kit utilizing equipment of a nature widely used andaccepted in a clinical environment.

It is a further object and feature of the subject invention to provide amethod for collecting cell samples on a filter media by drawing a liquidsample through the filter for trapping the cellular components of thesample in the media.

Other objects and features of the invention will be readily apparentfrom the accompanying drawings and detailed description of the preferredembodiment.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

FIG. 1 is a perspective view of a specimen collection apparatus of thesubject invention, particularly well suited for use in connection withmouthwash specimens.

FIG. 2 is a longitudinal cross-section of the specimen collectionapparatus shown in FIG. 1.

FIG. 3 is a view looking in the same direction as FIG. 2, showing thecollection apparatus after the specimen has been drawn through thefilter container for collecting on the filter media a sample containingthe cellular component of the specimen.

FIG. 4 is an enlarged, fragmentary view diagrammatically illustratingthe capture of the cellular component of the liquid specimen on thefilter media.

FIG. 5 illustrates the components of a typical kit embodying the presentinvention.

FIG. 6 is a cross section of another embodiment of the filter containerof the invention.

FIG. 7 is a cross-section like FIG. 6, showing the container separatedinto its mated halves to provide access to the filter media.

FIG. 8 is a cross section taken generally along line 8--8 of FIG. 7.

FIG. 9 is similar to FIG. 3, utilizing the filter container of FIGS. 6,7 and 8.

FIG. 10 illustrates a method for removing collected cells from thefilter media into an assay tube.

FIG. 11 illustrates an alternative embodiment of the filter container asshown in the kit assembly of FIG. 5, with the filter containerdisassembled to provide access to the filter media.

FIG. 12 is a plan view of the device of FIG. 11, showing the undersideof the top half.

FIG. 13 is a plan view of the device of FIG. 11, showing the topside ofthe bottom half.

FIG. 14 is an enlarged cross section taken generally along line 14--14of FIG. 11, but with the top and bottom halves in mated relationship.

FIGS. 15 and 16 show how the specific LCR probes used in examples 4 and5, respectively, align on their respective targets.

FIGS. 17 and 18 are photographs of strips on which DNA was tested aftercells were collected in accordance with the invention, and are describedin more detail in examples 4 and 5, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described in detail, including an explanationof the methods and theory as well as several varied embodiments thereofand working examples.

Part I: General Description

The collection apparatus of the invention is particularly well suitedfor collecting and archiving cells extracted from a liquid specimen foranalysis. In the present invention, a liquid "specimen" is distinguishedfrom a "sample" in that a "specimen" is the raw material collected fromthe patient, while a "sample" denotes a specimen that has been processedin some way; for example by concentrating sample cells by removal of thebulk of the liquid portion of the original specimen. Liquid specimensmay be obtained by invasive techniques, as in the case of blood orcerebrospinal fluid (CSF); or they may be non-invasively obtained, aswith urine, or rinses of various body parts or cavities, including butnot limited to vaginal douches, mouthwashes, and the like. A "rinse" or"lavage" refers to the use of a volume of liquid to wash over or througha body part or cavity, resulting in a mixture of liquid and cells fromthe body part or cavity.

A liquid specimen is considered a "high-volume" specimen when the amountof desired sample component is small relative to the quantity of liquidpresent. For example, urine and and mouthwashes are considered"high-volume" specimens since the expected cellular content (typicallybacterial and buccal cells, respectively) is small relative to thevolume of liquid obtained as the specimen. High volume specimens shouldbe distinguished from specimens such as culture swabs or centrifugepellets which are often resuspended in a limited amount of a diluentsolution.

Specimens may be collected from almost any source, including withoutlimitation, humans, animals, foods, the environment and the like. Theseliquid specimens are collected in a collection receptacle and drawntherefrom through a filter media which is selected to extract a cellularcomponent sample from the specimen. The cell specimen on the filtermedia may then be analyzed. The invention is designed to incorporatemethodology familiar to clinical technicians and relies on the used of apressure across the filter media to draw the specimen therethrough.

In general, the systems of the present invention comprise threecomponents: a collection receptacle, a filter media (generally enclosedin a housing) and a fluid collector tube.

A first embodiment of the collection apparatus is shown in FIGS. 1-4.The collection apparatus there shown is well-suited for use with acollection receptacle 10 having a slightly enlarged mouth 12 throughwhich a high-volume specimen such as an oral rinse or the like may bedeposited. It will be readily understood that other collectionreceptacles could be substituted for the receptacle 10, whereby thecollection apparatus of the subject invention could be used forcollecting and archiving other liquid specimens.

As best shown in FIGS. 1 and 2, the collection apparatus of the subjectinvention comprises a filter container 18 for supporting a filter media20 between an inlet port 24 and an outlet port 26. The inlet port 24 isadapted to communicate with the discharge port 14 of the collectionreceptacle, whereby a fluid specimen collected in the collectionreceptacle 10 may be discharged into the inlet port 24 and transferredthrough the filter 20 to the outlet port 26. As the fluid specimen flowsthrough the filter container 18, the cellular component of the specimenis captured and collected on the filter media to provide a cell sample.The cell sample captured on the filter 20 may then be archived and/ortransported for later analysis. It is an important feature of theinvention that the filter 20 may be dried and stored for easy transportto a clinical analysis facility.

In experimental tests, it has been found that any of a variety ofcommercially available filter elements produce suitable results incapturing high yield cell samples from a collected liquid specimen. Thefilter media will have a depth and pore size (or particle retentionsize) which is dictated primarily by the size of the cellular componentto be extracted and examined. It may be desirable to utilize compositefilter media of different ratings, particularly to assure efficientcollection of cells from a specimen that may contain mucous material. Inthe preferred embodiment of the invention, the filter is a bonded glassfiber depth filter such as the type commonly used as prefilters.

The physical properties of selected filter media are presented in thefollowing table:

                                      TABLE                                       __________________________________________________________________________    Source                                                                             Membrane   Particle retention (μ)                                                                Filtration rate*                                                                      Thickness (mm)                             __________________________________________________________________________    Millipore                                                                          AP15       0.2-0.6.sup.†                                                                     5.8     0.49 mm                                    Millipore                                                                          AP25       0.8-8.sup.†                                                                       3.2     0.92                                       Millipore                                                                          AP40       none given 3.9     0.41                                       Whatman                                                                            GF/A       1.6        13      0.25                                       Whatman                                                                            GF/B       1.0        5.5     0.68                                       Whatman                                                                            CF/C       1.2        10.5    0.2                                        Whatman                                                                            GF/D       2.7        16.5    0.68                                       Whatman                                                                            GF/F       0.7        6.0     0.42                                       Fisher                                                                             G8         2.5        0.5     0.69                                       Fisher                                                                             G2         2          4.8     0.66                                       Fisher                                                                             G6         1.5        1.6     0.32                                       Fisher                                                                             G4         1.2        3.2     0.28                                       Gelman                                                                             A/E Binder free                                                                          1          3.1     0.45                                       Gelman                                                                             Extra thick                                                                              1          7.4     1.3                                        Gelman                                                                             Metrigard  0.5        19.5    0.43                                       Poretics                                                                           Mixed ester cellulose                                                                    5          3.4     0.175                                      Poretics                                                                           Mixed ester cellulose                                                                    3          4.5     0.175                                      Poretics                                                                           Mixed ester cellulose                                                                    1.2        6.0     0.175                                      Poretics                                                                           Mixed ester cellulose                                                                    0.8        8.4     0.175                                      Poretics                                                                           Mixed ester cellulose                                                                    0.7        11      0.15                                       Poretics                                                                           Mixed ester cellulose                                                                    0.65       11.5    0.15                                       Poretics                                                                           Mixed ester cellulose                                                                    0.45       39      0.15                                       Poretics                                                                           Mixed ester cellulose                                                                    0.3        71      0.15                                       Poretics                                                                           Mixed ester cellulose                                                                    0.22       87      0.125                                      __________________________________________________________________________     *The manufacturers' specifications have been reduced to a common unit,        namely time in sec for 100 ml to pass through a 2.2 in diam filter at 1.9     psi.                                                                          .sup.† Although no retention size was given, the manufacturer          indicates that AP15 and AP25 are "good" as prefilters for filters having      the retention range listed.                                              

It can be seen from this table that the composition of the filter media20 is not crucial; it may be paper, glass fiber, esters of cellulose orother composition. What is more important is that a filter media isselected that has a retention size compatible with the approximate sizeof the cells to be filtered. Selecting the largest such suitable mediawill enable the fastest flow rates. From the above table, it is possiblefor the person skilled in the art to select a suitable filter mediabased on these criteria, provided the approximate size of the cells tobe filtered is known or can be determined.

Liquid specimens in the collection receptacle 10 are forced through thedischarge port 14, the inlet port 24 and the filter media 20 beforeexiting the filter container 18 via the outlet port 26. Preferably theoutlet port is connected to a disposal container 40 which collects thefiltrate which is to be discarded. The driving force for this movementof liquid is a pressure differential across the filter media. Althoughthe pressure differential may be created by positive pressure from abovethe filter 20, the preferred method utilizes a negative pressure orvacuum applied to the underside of the filter 20. This draws the liquidthrough the filter media as the cellular components of the specimen arecollected on the filter.

According to a most preferred configuration, the disposal vessel 40 alsoprovides the negative pressure on the underside of the filter. This canbe easily accomplished through the use of an evacuated vessel or tubewhich, upon connection to the outlet port 26, draws the liquid throughthe filter and into the disposal tube 40. Such an evacuated vessel neednot represent a complete vacuum but need only represent a sufficientlylow pressure to permit the drawing of a sufficient volume of liquidthrough the filter media. Thus, such a vessel is referred to herein as a"hypobaric" or "sub-atmospheric pressure" vessel or chamber. A suitableexample of a hypobaric vessel is the evacuated tube known asVacutainer™, produced and sold by Becton-Dickinson, Rutherford, N.J.

Preferably the hypobaric vessel includes a closure 38 such as a stopperor septum that can selectively be opened to the outlet port 24 of thefilter container 18. As an alternative, a valving means could be used asa selective closure.

Ideally the connections between the collection receptacle discharge portand the filter container inlet port, and between the filter containeroutlet and the means for generating a pressure differential across thefilter are "sealing engagements", by which is meant that the connectionis essentially air-tight. It is important that the connections beessentially airtight for proper operation. The reduced pressure at thefilter container outlet will draw liquid specimen through the filtermedia when the driving force, atmospheric pressure, is applied at theopen end of the collection receptacle on top of the fluid. If leaks orpoor seals permit atmospheric pressure to enter the system below thehead of liquid specimen, the flow and filtration will be inefficient.This caveat applies to filter container housings that are formed as twomated halves as well as to the connections between component parts ofthe apparatus.

The physical principles that govern the collection device can beunderstood in terms of the theory that describes pressure drivenfiltration. Application of a pressure gradient P to a filter mediumresults in a flow of liquid volume V per unit time t. The equation thatrelates these variables is:

    dV/dt=KP/u

where u is the liquid viscosity and K is the flow coefficient. In asystem where the pressure gradient is provided by an evacuated chamber,P may be presumed to equal one atmosphere. The flow coefficient K isequal to the inverse of the resistance, R, to liquid flow through themembrane; K=1/R.

In addition, the volume of specimen fluid passing through the collectionmembrane contains cells. As the cells are trapped in the membrane theresistance to fluid flow, R, increases. To simplify the model we assumethat the resistance to fluid flow changes in a linear fashion withmembrane loading, or K=Km (1-CV/W) where Km is the initial membranecoefficient, C is the number of cells per unit volume V, and W is themaximum number of cells the membrane can hold. In this approximation theflow coefficient starts off at Km and decreases to zero when the volumeof liquid passing through the membrane contains the maximum number ofcells that the membrane can hold. The phenomenon of increasing membraneresistance as a result of the filtration process is generally termed"membrane fouling". More accurate descriptions of membrane fouling canbe derived, however, they will not substantially change the qualitativedescription of the sample collection device of the invention.

Another feature of the present system is the use of a finite volumehypobaric vessel to provide the driving force for filtration of thespecimen. As the liquid specimen is pulled into the hypobaric vessel thepressure will change as predicted by the equation PV=nRT where P is thepressure, V is the vessel volume, n is the concentration of gas, R isthe gas constant the T is the temperature. The equation that describesfilling of a hypobaric vessel or Vacutainer™ tube is: ##EQU1## whereP_(o) is the initial pressure inside the vessel of volume V_(o) andP_(i) is the pressure inside the vessel after volume V of specimen hasbeen filtered. While it is not possible to create a perfect vacuum,every effort is made to create the best vacuum possible. For ourpurposes we will assume that P_(o) =0, i.e., we have a perfect vacuuminside the hypobaric vessel so that P_(i) remains constant and equal tozero during sample collection. For this reason, filtration in thespecimen collection device can be assumed to be carried out under theconstant pressure of 1 atmosphere.

The flow of liquid specimen is therefore described by the equation:##EQU2## whose solution is: ##EQU3## where S is a rate constant definedas ##EQU4##

Empirically, flow for a mouthwash specimen is observed to be initiallyvery rapid followed by a sharp drop in flow rate in about 30 secondsafter about 10 cc of liquid has been filtered. The long term behavior ofthe system is characterized by very slow flow or, for practicalpurposes, flow stops. Thus, the observed behavior is in completequalitative agreement with our simple exponential model. In addition, anempirical estimate of S may be obtained for a typical sample because itis generally accepted that long term behavior of an exponentiallyrelaxing system is obtained by 6×S; thus S=30/6=5 seconds.

From this simple model we can predict that the apparatus will collectapproximately the same number of cells independent of the starting cellconcentration. For example, if one sample filters 10 cc of specimen andstops in 30 seconds, a specimen with twice the cell concentration willfilter 5 cc in 15 seconds and both collected samples will contain thesame number of cells.

If a specimen does not contain a sufficient concentration of cells, themaximum loading of the membrane W will not be achieved due to the finitecapacity of the hypobaric vessel. However, this situation can be avoidedby providing a volume mark on the disposal vessel and discarding anyspecimen and associated sample that resulted in a filtered volumegreater than this value.

Of course in reality a perfect vacuum is not necessary or attainable.Typically, the vacuum is calibrated to inhale a defined volume. Thismeans that P_(o) does not actually equal zero, and that the drivingpressure gradient is not constant and approaches unity as the disposaltube acquires its defined volume of liquid. This fact complicates themodel equations somewhat, but does not qualitatively affect theconclusions.

Once the cellular component of the specimen is deposited on the filtermedium, the cell sample can be prepared for subsequent analysis. Boththe preparation steps and subsequent analysis steps may vary greatlydepending on the situation. For example, the filter media is typicallyremoved from the filter container 18 and the cell sample is generallydried onto the filter. After drying, the filter and its cell sample maybe stored and/or transported to an analysis site, either local orremote, for subsequent analysis. The filter media may be sectioned intoaliquots for a plurality of tests where desired.

Typical analyses performed on such cell samples include, withoutlimitation, DNA analysis, immunological analysis, direct staining forobservation by microscopic analysis. In some instances, the cells willbe removed from the filter by resolubilization in a buffer or diluent.The cells may optionally be treated with detergents, such as when celllysis is desired to release internal molecules or structures foranalysis. In cases of stubborn cells, such as plant cells andMycobacterium, the cells may require breakdown by physical, mechanicalor ultrasonic means. Typical DNA analyses include: standardhybridizations such as dot blot and the like, with or without prioramplification of signal or target. Signal amplification schemes usefulwith DNA analysis include multimer or chain branched labels, enzymelabels and the like. Target amplification schemes include LCR and PCR.Specific examples of analysis of the DNA molecules of cells collected inaccordance with the invention are provided in Part III, infra.

Alternatively, the cells may be treated with permeability enhancers,pore-forming agents, or other similar fixative agents, such as whenin-situ hybridization is the contemplated method for analysis. Suchanalysis methods are known in the art, as exemplified by, e.g. U.S. Pat.No. 5,225,326, issued Jul. 6, 1993 to Bresser, et al. In short, itshould be clear that virtually any analysis method may be employed oncells collected according to the invention.

An unique and surprising feature of the invention is the ability tomicroscopically observe cells directly on most filter media. The onlylimitation appears to be the intensity of light and, if sufficient,cells can be observed even on dried filter media. Visualization isenhanced, however, when the filter element is wetted with water prior toobservation.

Part II: Various Specific Embodiments

The general concepts described above are further illustrated in threespecific embodiments described here.

In the embodiment shown in FIGS. 1-4, the components are readilyavailable and familiar parts. For example, it has been found that a 20ml to 50 ml syringe barrel, with the needle removed, is a suitablecollection receptacle 10, although other collection receptacles may bereadily adapted to the method and apparatus of the invention in themanner well known to those skilled in the art. Ideally, the design andconfiguration of the collection receptacle 10 is governed by the volumeand source of the liquid specimen which it must collect. For instancethe open mouth 12 can be specially configured to facilitate closecontact with the source of the specimen so as to minimize any spillageor loss of specimen volume.

The discharge port 14 of the syringe barrel receptacle 10 is acylindrical, protruding male element having external threads at 16. Thethreads are designed to sealingly engage the inlet port 24 of the filtercontainer 18. The discharge port 14 may optionally include a removableseal or a stop cock or valve (illustrated at 15 in FIG. 9 in connectionwith another embodiment) so that the filter container 18 may beconnected to the collection receptacle after collection of the liquidspecimen without leakage of the specimen. Alternatively, the valve whenutilized may be placed directly on the filter container.

In the preferred embodiments, a filter container 18 is adapted forhousing and enveloping a filter media such as filter paper 20. Wheredesired, the filter paper 20 can be supported or retained in a filtersupport such as the peripheral or annular channel 22 defined by a pairof annular flanges provided in the inner sidewall of the container 18.As noted, the inlet port 24 on the container 18 is a female, internallythreaded receptacle adapted to be threadably secured to the dischargeport 14 of the receptacle 10. The means for connecting the receptacle 10to the filter container 18 is not critical, as long as the connectiondoes not leak. The threaded connection is used simply as a matter ofchoice, although other known mechanisms for sealing (e.g., Luer-Lok™ orbayonet type) are also within the scope of the invention.

The container 18 includes an outlet port 26 through which the filteredliquid specimen exits the filter container. In this embodiment, theoutlet port is an externally threaded cylindrical extension adapted forreceiving the base 28 of a typical hypodermic needle 30. The base 28 ofthe needle 30 is typically internally threaded for sealing engagementwith the port 26. As is also typical, the hypodermic needle 30 has ahollow bore 32, and is cut on a bias as shown at 34 to form a sharppoint at 36. In order to assure sterility and to seal the needle, athin, frangible membrane is typically provided over the opening at theend to close the tube channel 30.

The filter container 18 is preferably made of at least two sectionsadapted to be mated together such that the sections may be separated toremove the filter media 20 without destruction.

In this first embodiment, a 16 gauge hypodermic needle is attached tothe outlet port 26 of the container, for use in combination with a 14ml. Vacutainer™ hypobaric chamber which also serves as a disposal vessel40 for the spent specimen. In the embodiment shown, the vessel 40 isplaced in sealed communication with the outlet port 26 of the filtercontainer 18 by inserting the sharp tip 36 of the needle 30 into andthrough the sealing cap or stopper 38 of the vessel 40, as shown inFIGS. 2 and 3. When the needle is in sealed communication with thehypobaric interior of the vessel 40, a pressure gradient is createdacross the filter media 20 as was previously described. This draws theliquid specimen 39 from the collection receptacle 10 through the filtermedia 20 and ultimately into the disposal vessel 40 (see FIG. 3). Whileother means can be used to generate a flow of the liquid from thecollection receptacle, through the filter container and into thehypobaric chamber, the use of a hypobaric vessel such as a Vacutainer™type tube has a distinct advantage in that it provides a consistentmeasured initial force for drawing in a defined volume of fluid,providing consistent and predictable collection results.

The filter captures and collects the cellular component of the specimenas it passes therethrough to define a cell sample 42 (see FIG. 4). Thedischarged portion of the specimen is collected in the vessel 40 forsuitable disposal.

One of the advantages of the present invention is the ability to performmultiple analyses on the same sample. The designs of the following twoembodiments are intended to augment this advantage by avoiding orminimizing of physical manipulation or segmentation of the filtermaterial itself. One embodiment facilitates the punching out of discsfrom the filter while it is still enclosed in the housing; while anotherembodiment permits the collection on multiple separate filters which canthen be handled separately without any physical separation orsegmentation process.

A second embodiment of the collection apparatus is shown in FIG. 9, thefilter container being shown in greater detail in FIGS. 6-8. In thisembodiment the disposal vessel 40 is a hypobaric chamber essentially thesame as in the previous embodiment. The collection receptacle 80 isfunnel shaped and is particularly well suited for collecting urinespecimens and the like. It includes a discharge port 14 adapted tosealingly mate with the inlet port 24 of the filter container of thisembodiment. It should be understood that the various collectionreceptacles, the disposal vessels and the filter containers areesentially interchangeable between embodiments. One has only to ensurethat the discharge and inlet ports can sealingly engage, and that theoutlet port and hypobaric chamber can be selectively put into sealablecommunication, such as by means of a hypodermic needle 30 (see FIG. 9)which may then be inserted in a typical hypobaric device for drawing thefluid through the discharge port of the receptacle 80 and into thefilter container 58, in the manner previously described.

With reference to the filter container 58 shown in FIG. 6-8, thisembodiment of the filter container comprises a pair of mated sections 60and 62, adapted for sealable engagement in use. The upper section 60 (asshown) includes a filter support plate 70 for supporting an annularfilter 72 (see FIG. 8). The filter support 70 may preferably bedetachable from the upper housing section 60 for ease in inserting afilter medium. The annular filter 72 includes a central aperture 74which is adapted to be received in the raised boss 71 provided on thesupport plate 70 for properly seating the filter 72 relative to thesupport surface. The support plate 70 includes a plurality of apertures76 through which the liquid specimen can pass as it is drawn through thefilter container 58.

The lower container section 62 includes a disc shaped plate or support64 which abuts against the support 70 of upper section 60 when thecontainer 58 is assembled as shown in FIG. 6. The plate 60 includes aplurality of apertures 66 aligned with apertures 76 in the supportsurface 70. In a variation (shown in FIG. 10) the support plate 70 maybe integrally formed or fused with the support plate 60. A porous plug68 may be placed in the apertures 66, 76 to further direct flowtherethrough. This assures adequate contact between the filter media andthe liquid specimen for providing a sufficient capture of the cellularsample on the filter and can also serve to channel and concentrate thecollection of cells in discrete areas 77 on the filter 72 surroundingthe positions of the apertures 76. This discrete zone collection isillustrated in FIG. 8, which may be compared with FIG. 4, wherein thecells are collected "en masse" in a central area 42.

Discrete zones of collected cells provides two advantages. First, thezones are easier to divide or section into aliquots for performing aplurality of analyses. Second, the distribution of specimen flow todiscrete areas of the filter 72 may minimize and delay membrane fouling.This distribution or channeling may be accomplished by channels orapertures below the filter media (e.g. apertures 76), or by passages or"manifolds" leading up to the filter media (e.g. manifold 106 describedbelow in connection with FIGS. 11-14).

After collection of the cell sample 42, the halves 60, 62 of the filtercontainer 58 may be separated and the filter 72 removed. It may bedried, sectioned, transported and/or analyzed by any desired means, aswith the other filter 20.

A variant method for removing cell samples 42 from the filter container58 is shown in FIG. 10. In this variation the filter container 58includes a plurality of openings 84 around the perimeter of the upperwall 83 of the filter housing member 60. The openings 84 are in axialalignment with the apertures 66, 76 and the associated zones 77, and areinitially closed with a removable membrane or seal 81. After the cellsample 42 is collected in each of the various zones 77 on the filter,the bottom member 62 of the container 58 is removed. A suitablereceptacle such as vial 85 is then placed in communication with aselected zone 77 as shown in FIG. 10. Typically, the vial 85 willinclude an open mouth have an outer lip or rim 87 adapted to be insertedinto or connected with a respective aperture 76. A tool such as punch 86includes a tip 89 which is adapted to punch the associated cell samplezone 77 out of the disc and into the vial 85 where it can be stored foranalysis. Prior to using the punch 86, the seal 81 may be peeled awayfrom openings 84 in the upper housing member 60 or the punch may beadapted to puncture the seal 81 as well. This alternative method forremoving cell samples does not require that the entire filter beremovable from the housing

A third filter container embodiment is shown in FIGS. 11-14. As thereshown, a manifold filter container 88 comprises a pair of substantiallyrectangular sections members or halves 91 and 92. Each of these sectionscan be formed from a unitary mold with integral flow paths in the mannerwell-known to those skilled in the art. As shown in FIGS. 11 and 13, aninlet port 24 is provided in the nipple 100 extending outwardly from theright end of the lower section 92. An outlet port 26 is provided in theoutlet nipple 96 extending from the left end of upper section 91 (seeFIGS. 11 and 12). Longitudinal grooves 103 are molded into the uppersection 91 for defining the longitudinal lands or ribs 104. Similarlongitudinal grooves 107 are molded into lower section 92 for definingthe longitudinal lands or ribs 108. An integral through channel 102 isprovided in the nipple 96 and communicates the upper filter grooves 103with outlet port 26, via manifold 105. Likewise, an integral throughchannel 106 is provided in inlet nipple 100 and communicates the lowerfilter grooves 107 with the inlet port 24, via manifold 109. The ribs104 and 108 are preferably staggered (see FIG. 14) to create diagonalflowpaths through the filter media element 110 to maximize the area towhich the specimen is exposed as it is drawn through the filtercontainer, to maximize the cell yield in the captured cell sample. Inuse, the filter media element(s) 110 is placed in the longitudinalgrooves on the ribs 108 and is sandwiched between the upper and lowersections when assembled as shown in FIG. 14. The inlet nipple 96 isadapted to be secured to the discharge port of a suitable collectionreceptacle and the outlet nipple 100 is adapted to be secured to asuitable disposal vessel in the manner previously described.

In use, a filter media is placed in each groove and the halves aresealed together. Locking tabs or clamps (not shown) may be provided forsealingly engaging the container halves. A collection receptable issealingly engaged at the inlet end. When a pressure differential iscreated between the inlet port 24 and the outlet port 26 the liquid inthe collection receptacle flows through the integral channels defined bythe ribs and grooves, as shown in FIG. 14 to capture the cell samples.

One advantage of this embodiment is that more cells or particulates canbe filtered as the filter element 110 near the inlet becomes clogged byallowing the fluid specimen to flow through the grooves 103 until itreaches a less clogged portion of the filter media. It will be readilyunderstood by those who are skilled in the art that the particularconfiguration of the ribs and grooves is a matter of design choice andis largely dictated by the size of the cellular structure being capturedin the cell sample.

As is particularly shown in FIG. 5, the collection apparatus of theinvention can be assembled in kit form for ready use in a clinicalenvironment. The kit typically includes a collection receptacle 10, oneor more filter containers 58, 88, a hypobaric vessel 40 with a seal suchas cap 38, and means for applying the pressure differential of vessel 40to the outlet port 26 of the filter container. The means for applyingpressure may be the needle assembly 30 which is adapted to be attachedto the discharge end 26 of the respective filter containers and topuncute the seal 38 of the vessel 40. The component filter container 58,88, collector receptacle 10, needle assembly 30 and hypobaric chamber 40are all described in detail above.

Part III: Examples

Examples were run under laboratory conditions and demonstrate thefunctionality of the invention as disclosed herein. The followingabbreviations are used consistently with respect to the examples:

    ______________________________________                                        BSA:  Bovine serum albiumin                                                   CF:   Cystic fibrosis                                                         CFTR: Cystic fibrosis transmembrane regulator                                 EPPS: a buffer comprising N-(2-hydroxyethyl)piperazine-N'-(3-                       propane sulfonic acid)                                                  oligos:                                                                             Oligonucleotides, generally oligo-2-deoxyribonucleotides                NAD:  Nicotine adenine dinucleotide                                           TRIS: a buffer comprising tris(hydroxymethyl)aminomethane                     units:                                                                              a measure of the concentration of enzyme. Units for                           polymerase are as expressed by the manufacturer; units                        for ligase are defined such that 1 mg of 95% purified                         DNA ligase has a specific activity of about 1 × 10.sup.8                units.                                                                  ______________________________________                                    

Example 1: General Cell Collection Device

Filtration-Collection devices according to the present invention wereassembled as follows: Millipore (Millipore Corporation, Bedford, Mass.)AP25, 1 inch diameter glass fiber pre-filters were assembled into 25 mmSwinnex Disc Filter Holders (Millipore, Bedford, Mass.). These were thenattached to a 20 or 50 ml standard syringe barrel via the Luer entryport. Hollow bore hypodermic needles, 16 gauge, 1 inch long wereattached to the Luer fitting at the exit port. Samples are collected(see examples below for details) in the syringe barrel which acts as aconvenient funnel and the filtration is achieved by inserting the needlethrough the rubber stopper of a Vacutainer™ tube (Becton Dickinson,Rutherford, N.J.) and performing the filtration. When the filtrationprocess terminates, the excess liquid sample is discarded, the devicedissembled and the filter processed as in following examples.

Example 2: Specimen Collection:

A. Water Rinse: Each individual took 10 ml of drinking water and rinsedit a few times in their mouths to constitute the liquid specimen, andthen expectorated the specimen into the opening of a collectionreceptacle. A Vacutainer™ vessel was connected to apply a pressuregradient and the cell sample was collected on the filters as describedabove. After collection, the filters were removed from the collectiondevice and air dried on a lab bench. Dried filters were either processedor stored in a plastic bag at room temperature for up to 47 days untilprocessing.

B. Antiseptic Mouthwash Rinse: Samples were collected as in part A,except the individuals used 10 ml of commercially available antisepticmouthwash (Scope®, Proctor & Gamble, Cincinnati, Ohio) to constitute theliquid specimen. The AP40 filters disintegrated after collection andsample processing (see below).

Example 3: Processing of Collected Cell Samples:

A. Some of the collected filters were placed in a 1.7 ml Microcentrifugetube and 0.5 ml of sterile HPLC water was added. The sample tube wasthen incubated in boiling water for 20 min. and then cooled to roomtemperature. The liquid was removed to a new tube after a quick spin.

B. Other samples were collected as above, followed by an alternative KOHextraction. These sample filters were processed with 0.5 ml of 50 mMKOH. After incubation in boiling water for 20 min. 100 μL of 0.1 MTris-HCl, pH 7.5, was added to neutralize the solution. The solution wasthen centrifuged for 20 min at 14,000 rpm and the supernatant wastransferred to a new tube.

Example 4: LCR® Amplification and Detection of CFTR gene

A. Amplification: LCR® amplification was performed using the so-called"double-gap" strategy as described by Backman, et al. in European PatentApplication 0 439 182 (1991). Four probes (SEQ ID NOS. 1, 2, 3 and 4)were synthesized and labeled with biotin or fluorescein haptens as shownbelow using standard techniques known in the art.

    __________________________________________________________________________                                       SEQ. ID No.                                __________________________________________________________________________    Probes #1.                                                                          5'-fluorescein-GGCACCATTAAAGAAAATATCA-3'                                                                   1                                          Probes #2.                                                                          5'-p-GATATTTTCTTTAATGGTGCC-3'                                                                              2                                          Probes #3.                                                                          5'-p-GGTGTTTCCTATGATGAATATAG-biotin-3'                                                                     3                                          Probes #4.                                                                          5'-biotin-CTATATTCATCATAGGAAACACCAAAGA-3'                                                                  4                                          __________________________________________________________________________

The alignment of these probes with exon 10 of the CFTR gene (SEQ ID No.5) is shown in FIG. 15. The target is region 1612-1690 as numbered byZielenski et al., Genomics 10, 214-228 (1991).

LCR was carried out for 40 cycles with each of the four probes (SEQ IDNOS. 1, 2, 3 and 4) in a total volume of 100 μL with the following finalconcentrations: 50 mM EPPS pH 7.8, 20 mM potassium (added as KOH toadjust the pH of the buffer and as KCl to achieve 20 mM K+), 30 mMMgCl₂, 100 μM NAD, 10 μM nucleoside triphosphates dCTP and dTTP, 100 μMof each of the 4 probes, 3 units of DNA polymerase (Amplitaq®,Perkin-Elmer/Cetus, Emeryville, Calif.), and 3400 units of Thermusthermophilus DNA ligase. 10 μL of processed cell sample from Example 3Awas used for each amplification. Human placental DNA (50 ng) was used ascontrol to monitor the amplification process. The LCR® process itselfoccurred in a model 480 Thermal Cycler (Perkin-Elmer, Norwalk, Conn.)using a denature profile of 95° C. for 3 minutes followed by a cyclingprofile of 85° C. for 30 sec and 57° C. for 60 sec.

B. Detection by Immunochromatography: Antisera to adamantane,fluorescein, and biotin were raised in rabbits against adamantane-BSA,fluorescein-BSA or biotin-BSA. These antisera were purified by passagethrough protein A Sepharose® or protein G Sepharose® (Pharmacia,Piscataway, N.J.) and diluted in 0.1M TRIS pH 7.8, 0.9% NaCl, 0.1% BSA,1% sucrose, and a trace of phenol red. Portions (0.2 μL) of thesediluted anti-adamantane and anti-fluorescein antisera were jetted onto7.3×40 mm strips of nitrocellulose (AE98, 5 μm, Schleicher and Schuell,Dassel, Germany).

Anti-biotin antiserum was conjugated to polystyrene uniformly-dyed bluelatex particles (Bangs Laboratories, Carmel, Ind.). Particles (380 nmdiameter) were diluted 1.25 in water to give 1 ml at 0.4% solids and 10μL of anti-biotin at 1 mg/ml was added. The suspension was mixed on avortex mixer for 45 seconds, and 5 μL of 5% casein in 0.1M TRIS pH 7.8was added.

For detection, 21 μL of the anti-biotin conjugate (blue latex) wasdiluted with 16 μL buffer (0.1M TRIS pH 7.8 0.9% NaCl, 3% alkali-treatedcasein), and mixed with 5 μL of LCR® amplification product. Anitrocellulose strip containing anti-fluorescein or anti-adamantane, orboth, was introduced to the conjugate suspension, and chromatography wasallowed to proceed for five minutes, substantially as described inpublished application EP 0 357 011 A2. The strip was dried and is shownin FIG. 17. The presence of a colored spot at the locus ofanti-fluorescein application indicated the presence of a specific LCR®product.

FIG. 17 shows sample strips processed from different individuals atdifferent times following collection on filters as follows: Strip C. ispurified placental DNA. Strips 1-5 are samples from 5 differentindividuals processed from filters 47 days after collection (strip 1),45 days after collection (strip 2), and one day after collection (3individuals, strips 3, 4 and 5); and strip B is a blank control, with noDNA added.

Example 5: LCR® Amplification and Detection of β globin gene

A. Amplification: LCR® amplification was performed using the so-called"double-gap" strategy as described by Backman, et al. in European PatentApplication 0 439 182 (1991). Four probes (SEQ ID NOS. 6, 7, 8 and 9)were synthesized and labeled with biotin or adamantane haptens as shownbelow using standard techniques known in the art. The alignment of theseprobes on the β- globin gene (SEQ ID No. 10) is shown in FIG. 16.

    __________________________________________________________________________                                     SEQ ID No.                                   __________________________________________________________________________    Probe 1.                                                                           5'adamantane-CCGAGGGCCTCACCACCAACTTCA-3'                                                                  6                                            Probe 2.                                                                           5'-p-GAAGTTGGTGGTGAGGCCCTGGG-3'                                                                           7                                            Probe 3.                                                                           5'-p-CCACGTTCACCTTGCCCCACAGG-biotin-3'                                                                    8                                            Probe 4.                                                                           5'-biotin-CCTGTGGGGCAAGGTGAACGTGGA-3'                                                                     9                                            __________________________________________________________________________

LCR® amplification was carried out as in Example 4A except that onlydTTP is needed to fill the gap in these probes; and 10 μL cell samplesprocessed with both water and KOH extractions were used (see Examples 3Aand 3B).

B. Detection by Immunochromatography: Immunochromatographic strips areprepared as in Example 4B. Detection of amplified products was performedas described above except the following conjugate was used.

Anti-biotin antiserum was conjugated to polystyrene uniformly-dyed bluelatex particles (Molecular Probes, Inc. Eugene, Oreg.). Particles (306nm diameter) were diluted in 1/40 in water to give 5 ml at 0.05% solids.and 50 μL of anti-biotin at 1 mg/ml was added. The suspension was mixedfor 5 min. 50 μL of 5% casein in 0.1M TRIS pH 7.8 was then added andmixed for 15 min. The solution was transferred to 1.7 ml microcentrifugeand centrifuged for 10 min 16 10,000 rpm. The supernatant was thenremoved. Pellet from original 1 ml solution was resuspended with 980 mlof HPLC grade water and all five ml were combined.

Data from this example is shown in FIG. 18, wherein the followingannotations are used: Strip C. is purified placental DNA (50 ng); Strips1 to 3 are from filters processed by water extraction per example 3A;Strips 4 and 5 are from filters processed by KOH extraction per example3B with the addition of spiked-in pure placental DNA (50 ng).

From these experiments and others (data not shown) it has been observedthat conventional KOH extraction can be used for blunt LCR, but is notsuitable for Gap LCR (note the absence of signal in strips 4 and 5 ofFIG. 18. Apparently, the KOH extraction protocol causes inhibitors ofAmplitaq® DNA polymerase to be co-extracted. In the case of Gap LCR, thewater extraction alternative protocol is preferred.

It is also observed that tap water appears to be better than Scope®mouthwash for obtaining viable cells. Experience with Scope® shows thatthe filter disintegrated completely, perhaps due to the alcohol contentof this commercial product.

While certain features and embodiments have been described in detailherein, it will be readily understood that the invention may includeother modifications and enhancements. The invention for which protectionis sought is defined by the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 10                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (synthetic)                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GGCACCATTAAAGAAAATATCA22                                                      (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (synthetic)                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GATATTTTCTTTAATGGTGCC21                                                       (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (synthetic)                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GGTGTTTCCTATGATGAATATAG23                                                     (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (synthetic)                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CTATATTCATCATAGGAAACACCAAAGA28                                                (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 68 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: genomic DNA                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TATGCCTGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATGATGAATATAGATA60                CAGAAGCG68                                                                    (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (synthetic)                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CCGAGGGCCTCACCACCAACTTCA24                                                    (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (synthetic)                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GAAGTTGGTGGTGAGGCCCTGGG23                                                     (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (synthetic)                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       CCACGTTCACCTTGCCCCACAGG23                                                     (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (synthetic)                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CCTGTGGGGCAAGGTGAACGTGGA24                                                    (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: genomic DNA                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CCAGGGCCTCACCACCAACTTCATCCACGTTCACCTTGCCCCACAGG47                             __________________________________________________________________________

What is claimed is:
 1. A method for collecting on a filter media cellsfrom a liquid specimen having a cellular component, the methodcomprising the steps of:depositing a liquid specimen suspected of havinga cellular component into an open end of a collection receptacle havinga discharge port; placing the discharge port in fluid communication witha filter media by means of a sealed passage therebetween, the filtermedia having a pore size sufficiently small to retain desired suspectedcells from the liquid specimen,. wherein the filter media is housedbetween an inlet port and an outlet port of a filter container, theinlet port being adapted for sealably engaging the discharge port; andapplying a pressure differential across the filter media and extendingto the liquid specimen by means of the sealed passage to force theliquid specimen through the discharge port of the collection receptacleand through the filter media, wherein said differential pressure isapplied by puncturing a penetrable closure element of a closed hypobaricchamber with said outlet port in order to put said outlet port andclosed hypobaric chamber in sealed fluid communication, thereby placingthe filter container under a relative vacuum and capturing on the filtermedia a sample of any cellular component of the liquid specimen.
 2. Themethod of claim 1, including the further steps of removing at least someof the filter media from the filter container and drying the samplecollected on the filter media.
 3. The method of claim 2, comprising astep of segmenting the filter media and captured sample into discretesections for separate analysis.
 4. The method of claim 3, wherein thefilter container includes means for distributing the liquid specimen todiscrete areas of the filter media.
 5. The method of claim 2, includingone or more further steps selected from storing or transporting a sampledried on said filter media.
 6. The method of claim 1, including afurther step of processing the sample for analysis of the presence oramount of a selected constituent.
 7. The method of claim 6, wherein theselected constituent is a segment of nucleic acid.
 8. The method ofclaim 7, wherein the nucleic acid segment is amplified prior to saidanalysis.
 9. The method of claim 1, wherein said liquid specimen is ahigh-volume specimen.
 10. The method of claim 9, wherein said liquidspecimen is a mouthwash specimen.
 11. The method of claim 1, whereinsaid puncturing includes mounting a hollow bore needle to said outletport and puncturing the penetrable closure element with said needle. 12.A cell collection apparatus for collecting, archiving and/ortransporting a cell sample generated from a liquid specimen,comprising:a collection receptacle having an open end for receiving aliquid specimen and a discharge port through which said liquid specimencan leave the collection receptacle; a filter container having an inletport adapted for sealably engaging the discharge port of said collectionreceptacle, an outlet port through which said liquid specimen can leavethe filter container and a filter media supported in the filtercontainer between said inlet and outlet ports, whereby the liquidspecimen entering said filter container via the inlet port passesthrough said filter media before leaving via said outlet port, saidfilter media being adapted for receiving and capturing a cellularcomponent of the liquid specimen for defining a cell sample for analysisand said outlet port being adapted for sealably puncturing a rupturableseal; and a hypobaric vessel having a rupturable seal, wherebypuncturing said rupturable seal with said outlet port places them insealed communication such that a relative vacuum is applied to theoutlet port thereby drawing the liquid specimen through the filtermedia.
 13. The cell collection apparatus of claim 12, wherein saidhypobaric vessel serves as a discharge vessel for receiving the liquidspecimen passing through the filter media and the outlet port of thefilter container.
 14. The cell collection apparatus of claim 12, whereinthe filter media is a discrete element, all or part of which isremovable from said filter container.
 15. The cell collection apparatusof claim 12 wherein the filter container comprises a pair of matedmembers, the first member including a filter media support adapted forretaining and seating the filter media, and the second member adapted tobe placed in mated assembly with the first member to form an assembledcontainer for releasably housing the filter media.
 16. The cellcollection apparatus of claim 12, further comprising a filter supportmeans for supporting the filter media.
 17. The cell collection apparatusof claim 16, wherein said filter support means comprises a plate havinga plurality of apertures for localizing collected cells in discreteareas corresponding to said apertures.
 18. The cell collection apparatusof claim 17, wherein said filter container defines holes in registrationwith the apertures in said plate, said holes and apertures being adaptedfor receiving a punch to punch out and remove sections of the cellsample from the filter container.
 19. The cell collection apparatus ofclaim 12, further comprising channeling means for distributing theliquid specimen to discrete areas of the filter media or to distinctfilters.
 20. The apparatus of claim 12, further including a hollow boreneedle adapted to be secured to said outlet port for sealably puncturingthe rupturable seal of said hypobaric vessel.
 21. A cell samplecollection kit for collecting and obtaining for analysis a cell samplefrom a liquid specimen, the kit comprising:a filter container having aninlet port and an outlet port; a filter media removably housed in thefilter container between the inlet and outlet ports; a collectionreceptacle having an open end for receiving a liquid specimen and adischarge port adapted to be selectively mounted in communication withthe inlet port of the filter container; a hypobaric chamber adapted tobe placed in communication with the outlet port of the filter containerfor generating a pressure differential across the filter media fordrawing the liquid specimen from the collection receptacle and throughthe filter media for capturing a cellular component of the liquidspecimen on the filter media; and a hollow bore needle adapted to bemounted on the outlet port of the filter container, the needle borebeing in communication with the outlet port of the filter container whenmounted thereon, the hypobaric chamber including a closure adapted to bepunctured by the tip of the needle for drawing the liquid specimen fromthe collection receptacle into the filter container and through thefilter media for discharge through the outlet port into the hypobaricchamber.
 22. The kit of claim 21, wherein the filter media is supportedwithin the filter container by a supporting ridge or plate.
 23. The kitof claim 21, wherein the filter container further includes channel meansfor distributing the liquid sample to discrete areas of said filtermedia.
 24. The kit of claim 21, wherein said filter media is made of amaterial selected from the group consisting of glass fiber, paper andcellulose esters.